research communications

Acta Cryst. (2017). F73, 657–663 https://doi.org/10.1107/S2053230X17015941 657

Received 31 August 2017

Accepted 1 November 2017

Edited by N. Strater, University of Leipzig,

Germany

Keywords: fluorescence; trace fluorescent

labeling; low-cost imaging; protein

crystallization.

A low-cost method for visible fluorescence imaging

Crissy L. Tarver and Marc Pusey*

Department of Biological Science, University of Alabama in Huntsville, Huntsville, AL 35899, USA. *Correspondence

e-mail: puseym@uah.edu

A wide variety of crystallization solutions are screened to establish conditions

that promote the growth of a diffraction-quality crystal. Screening these

conditions requires the assessment of many crystallization plates for the

presence of crystals. Automated systems for screening and imaging are very

expensive. A simple approach to imaging trace fluorescently labeled protein

crystals in crystallization plates has been devised, and can be implemented at a

cost as low as $50. The proteins �-lactoglobulin B, trypsin and purified

concanavalin A (ConA) were trace fluorescently labeled using three different

fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and

Pacific Blue (PB). A crystallization screening plate was set up using

�-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled

with each probe, and a mixture consisting of 50% PB-labeled ConA and 50%

CR-labeled ConA. The wells of these plates were imaged using a commercially

available macro-imaging lens attachment for smart devices that have a camera.

Several types of macro lens attachments were tested with smartphones and

tablets. Images with the highest quality were obtained with an iPhone 6S and an

AUKEY Ora 10� macro lens. Depending upon the fluorescent probe employed

and its Stokes shift, a light-emitting diode or a laser diode was used for

excitation. An emission filter was used for the imaging of protein crystals labeled

with CR and crystals with two-color fluorescence. This approach can also be

used with microscopy systems commonly used to observe crystallization plates.

1. Introduction

The structure and function of many proteins are often deter-

mined by X-ray crystallography, which requires crystals with

high regularity. Crystallization conditions that facilitate the

growth of a single crystal that diffracts to atomic resolution

must be ascertained by screening an assortment of solutions.

Screening conditions involves setting up multiple plates of

trial conditions and then periodically reviewing these condi-

tions for the growth of crystals. The reviewing process may be

carried out manually using a microscope, or it may be

performed using an automated plate-imaging system. There

are two major difficulties in the plate-reviewing process:

distinguishing macromolecule crystals from salt crystals and

the actual detection of macromolecule crystals. Automated

imaging systems address these difficulties by focusing the

detection approach on characteristics that are likely to be

present in macromolecules but not small molecules, such as

the presence of tryptophan. Imaging approaches based on

recognizing crystals, typically an extensive process, have some

of the same difficulties as direct visual observation. Most

recently developed methods now use fluorescence or some

other optical manipulation to clearly reveal the presence of

macromolecule crystals.

ISSN 2053-230X

An early approach was to make use of the intrinsic fluor-

escence in macromolecules (Judge et al., 2005; Dierks et al.,

2010) arising primarily from the presence of the amino acid

tryptophan. The use of UV is not recommended for direct

microscopy visualization. Two-photon fluorescence has been

used with an excitation wavelength of 515 nm (Madden et al.,

2011). This method requires scanning a high-intensity light

source through a region of interest, using a high numerical

aperture lens to achieve a sufficiently high photon density that

fluorescing molecules can absorb two photons, at twice the

wavelength of the normal absorption, within 10�15–10�16 s of

each other (Xu & Webb, 1997). This requires a high-power

light source plus a means of scanning the light through the

sample and acquiring the emission signal from each scanned

point. Another approach is the use of second-order nonlinear

imaging of chiral crystals, also known as SONICC (Kissick et

al., 2010). A different fluorescence approach is based upon

visible-light excitation of native fluorescence (Lukk et al.,

2016). The fluorescence is apparently not chromophore-

dependent, but is believed to be based on conjugated double

bonds and electron delocalization from crystal packing. The

fluorescence can be obtained over a range of visible wave-

lengths, but was found to be strongest with excitation at

<405 nm, with a rather wide Stokes shift (excitation at

�405 nm, emission at �485 nm). The fluorescence dramati-

cally increased with decreasing temperature and varied from

protein to protein. Some proteins were found to not fluoresce.

A relatively low-cost approach (<1000 Euro) has been

described by Watts et al. (2010). This approach is based on the

increase in fluorescence when a soluble probe, in this case

1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), binds to

hydrophobic regions, as described by Groves et al. (2007).

The trace fluorescent labeling (TFL) approach was, as with

the above methods, introduced to speed up and simplify the

process of finding macromolecule crystals in screening plates

and to differentiate macromolecule crystals from salt crystals

(Forsythe et al., 2006). Relatively simple instrumentation can

be used, and the technique is not dependent upon the

presence of fluorescing amino acids as the fluorescent probe is

covalently bound to the protein. The safety issues associated

with UV fluorescence are avoided, and one can choose from a

range of colors when using visible fluorescence. The labeling

method is simple and takes about 15 min to carry out, and the

target labeling level of 0.1–0.5% is achieved if it is performed

according to protocol (Pusey et al., 2015). Labeling at this level

has been shown to not affect the nucleation rate or the

diffraction quality of the crystals obtained (Forsythe et al.,

2006).

The primary barrier to the use of all fluorescence-based

methods is the cost of the required instrumentation. Relatively

low-cost fluorescence microscopy systems can be obtained;

however, they are more suited for looking at slide-mounted

specimens instead of crystals in thicker and larger crystal-

lization plates. While fluorescence may be the most unam-

biguous means of identifying macromolecule crystals and

crystallization conditions, it is not readily available to smaller

structural biology research groups or students. Here, we

present a simple and low-cost approach to visible fluorescence

imaging of TFL crystallization screening plates.

2. Materials and methods

2.1. Proteins

The proteins used throughout this work were trypsin

(Sigma, catalog No. T1426), �-lactoglobulin B (Sigma, catalog

No. L8006) and concanavalin A (ConA). ConA was purified

in-house from jack beans using the method of Agrawal &

Goldstein (1967). The purified ConA was stored in crystalline

form at 4�C. An aliquot of the precipitate was removed,

concentrated by centrifugation, dissolved in weak ammonium

hydroxide solution and underwent buffer exchange using a

centrifugal desalting column into the crystallization buffer

0.025 M Na HEPES, 0.025 M NaCl pH 7.5.

2.2. Crystallization

Each protein was trace fluorescently labeled (TFL) as

described previously (Pusey et al., 2015) using either Cascade

Yellow (CY; Invitrogen, catalog No. C-10164), Carboxy-

rhodamine 6G (CR; Invitrogen, catalog No. C-6157) or Pacific

Blue (PB; Invitrogen, catalog No. P-10163). Crystallization

plates were set up using (i) CY-labeled ConA, (ii) CR-labeled

ConA, (iii) PB-labeled ConA, (iv) a mixture containing 50%

research communications

658 Tarver & Pusey � A low-cost method for visible fluorescence imaging Acta Cryst. (2017). F73, 657–663

Figure 1Top: diagram of the cell-phone-based fluorescence imaging system. (a)Cell phone; (b) emission filter; (c) 10�macro lens with attached hood; (d)LED mounted in lens hood. Not shown are the aluminium tube used tohold and aim the LED, the LED power supply and the clip that holds theassembly to the cell phone. Bottom: image of the complete cell-phone-based system, showing the macro lens with black hood attached to aSamsung Galaxy cell phone. Also shown are the power supply and theconnections to the LED.

PB-labeled ConA and 50% CR-labeled ConA, (v) CR-labeled

�-lactoglobulin B and (vi) CY-labeled trypsin. The sitting-

drop vapor-diffusion method was used for all crystallizations.

ConA was crystallized using the JCSG-plus screen (Molecular

Dimensions, catalog No. MD1-37) in Corning CrystalEX

plates (Hampton Research, catalog No. HR8-140). Each of the

96 reservoirs was filled with 50 ml precipitant solution, and the

three wells were prepared at protein:precipitant ratios of 1:1,

2:1 and 1:2. All plates were stored at room temperature.

Trypsin was crystallized using the conditions given on the

Rigaku website (http://www.rigaku.com) and �-lactoglobulin

B was crystallized from 0.1 M sodium citrate, 3.0 M ammo-

nium sulfate pH 4.0.

2.3. Fluorescent imaging

The imaging lens used was an AUKEY Ora 10� macro lens

purchased from Amazon (Fig. 1). The lens clips onto the

camera of a smartphone or tablet, and it comes with a hood.

The hood was modified by epoxying a short piece of 5 mm

internal diameter aluminium tubing to a hole drilled in the top

of the hood, near to where it attaches to the lens assembly.

When applying the epoxy, the aluminium tubing was aimed to

the center of the hood by a second piece of aluminium tubing

with a 5 mm outer diameter to indicate the path of the exci-

tation beam. After the epoxy had set, the exterior of the hood

was covered with a coating of flat black paint. The excitation

source used to screen crystallization plates containing protein

labeled with Cascade Yellow or Pacific Blue was a 5 mm light-

emitting diode (LED; LED Supply, catalog No. L3-0-

U5TH15-1; Fig. 2). This LED has a peak wavelength of 400 nm

and an emission half-angle of �15�. Power to the LED was

supplied by a 5 V power supply through a DynaOhm DC

resistor module (LED Supply, catalog No. 04006–020) to

control the LED current to 20 mA. The LED plugs into a

connector and can be easily changed for one with a different

wavelength. Alternatively, a 532 nm diode laser was used for

excitation of protein crystals labeled with Carboxyrhodamine

6G. The monochromatic laser light was launched into a fiber

optic to bring it to the hood, where a cone of light was emitted

onto the imaging area. An emission filter suitable for the

fluorescent probe to be imaged was inserted between the

camera and the clip-on lens.

3. Results and discussion

3.1. Magnification lenses

Several commercially available clip-on macro lenses for

smart devices were obtained and evaluated for fluorescent

imaging applications. Images with the best overall quality for

the cost were obtained using an AUKEY Ora lens with an

iPhone 6S, and this lens provided a convenient means of

mounting an excitation light source. Modifications to accom-

modate an excitation source were simple to make. One source

of difficulty in using these clip-on lenses was stabilizing them

firmly over the center of the camera lens. The more expensive

Olloclip Macro Pro Lens for iPhones eliminated the stability

research communications

Acta Cryst. (2017). F73, 657–663 Tarver & Pusey � A low-cost method for visible fluorescence imaging 659

Figure 2Complete LED power supply and the components used in assembling this light source. The LED bulb, the wiring connector that the LED plugs into andthe LED current-limiting resistor are labelled A, B and C, respectively, and the individual components are shown in (a), (b) and (c).

difficulties, but introduced new difficulties with the placement

of an emission filter.

3.2. Fluorescent probes

This imaging approach was tested using several fluorescent

probes. Crystallization plates containing ConA labeled with

PB (excitation and emission at 404 and 455 nm, respectively)

were initially imaged through a bandpass filter (Edmund

Optics, catalog No. 65-626). Subsequent experiments indicated

that the filter was not necessary, resulting in this system having

the fewest components and the lowest cost. Protein crystals of

ConA TFL with PB were excited with a 400 nm blue LED and

imaged without the use of an emission filter (Fig. 3a). These

crystals failed to excite under the 532 nm green diode laser

(Fig. 3b).

The original implementation of this system used CR as the

fluorescent probe. The Stokes shift for CR is �28 nm (exci-

tation at �524 nm, emission at �552 nm), the LED peak

wavelength was �525 nm with a full-width at half-maximum

(FWHM) wavelength range of �30 nm, and the addition of a

550 nm high-pass emission filter (Edmund Optics, catalog No.

62-977) between the macro lens and the camera lens was

required. The wavelength spread of the LED resulted in a

significant amount of excitation light still passing through the

filter, which obscured the fluorescent signal. However, protein

crystals of CR-labeled ConA were detected and imaged using

a 550 nm high-pass emission filter and a 532 nm diode laser as

an excitation source (Fig. 3c). The monochromatic laser light

was launched into a fiber optic to bring it to the optical port,

where a cone of light was emitted onto the imaging area.

These crystals failed to fluoresce with the 400 nm blue LED

(Fig. 3d).

Use of this approach is not limited to the fluorescent probes

mentioned. LEDs are available with emission peaks suffi-

ciently close to those of most fluorescent probes. Fluorescence

visualization is simplified when using probes having a larger

Stokes shift, but with some forethought those having a rela-

tively narrow shift can be accommodated, as shown in this

work. One important consideration is that fluorescent probes

are often very sensitive to their local environment and may

undergo absorption and/or emission shifts as a result.

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660 Tarver & Pusey � A low-cost method for visible fluorescence imaging Acta Cryst. (2017). F73, 657–663

Figure 3(a) Crystals of ConA labeled with the fluorescent probe PB and excited using a 400 nm blue LED without using an emission filter. (b) The well shown in(a) imaged using a 532 nm green laser diode and a 550 nm high-pass emission filter. (c) CR-labeled ConA crystals imaged using a 532 nm green laserdiode and a 550 nm high-pass emission filter. (d) The well shown in (c) imaged under the light from a 400 nm blue LED without an emission filter.

However, this may be mitigated by the fact that the probe will

be buried in the relatively protected environment of the

crystal, potentially precluding effects originating from the

bulk solution.

3.3. Emission filters

In the case where the Stokes shift of the fluorescent probe

was very narrow, emission filters were needed to prevent

reflected excitation light from overwhelming the fluorescent

signal. The filters needed to have a very sharp cutoff and a

very high blocking optical density at nontransmission wave-

lengths to minimize reflections obscuring the fluorescence

emission. However, the use of a filter more than quadrupled

the cost of the system. Two variations of a laser-excitation

approach were tried: manually directing a green laser pointer

through the translucent hood around the lens and directing

the beam into a fiber optic that was passed through a close-

fitting aluminium tube through the hood. In both cases the

550 nm high-pass filter was required to block excitation

reflections from the camera. While both approaches work, it

was felt that switching to a diode laser-excitation source

detracted from the goal of an inherently simple low-cost

imaging system. However, use of a laser-excitation source does

facilitate imaging fluorescence from probes having a very

small Stokes shift.

Although some excitation light enters the camera with the

filter-free approach used with PB, it is somewhat attenuated by

the glass optics of the macro lens and it is readily differ-

entiated from the emission signal. PB has a sufficiently wide

Stokes shift that an emission filter was not needed, although

one could be used to further enhance the fluorescence over

the background. The fluorescent probe CY (excitation at

�410 nm, emission at �560 nm) can also be excited using a

400 nm LED. Owing to the similarities between the CR and

CY emission peaks, the 550 nm high-pass filter used with CR

was tested with CY (results not shown). As with PB, the use of

an emission filter was not necessary for imaging protein

crystals labeled with CY. One negative aspect of the use of CY

was the fact it is no longer commercially available. The

fluorescent probe Pacific Orange (PO; Invitrogen, catalog No.

P-30253; excitation at �405 nm, emission at �551 nm) is

commercially available and could be used with the same

excitation LED.

3.4. Two-color fluorescence

The method can also be used for two-color fluorescence,

which is useful for the visualization and imaging of complexes.

This approach required the use of an emission filter. Crystal-

lization screening plates were set up using (i) ConA labeled

with PB, (ii) ConA labeled with CR and (iii) a mixture

containing 50% PB-labeled ConA and 50% CR-labeled

ConA. Screening plates containing CR-labeled ConA were

imaged using a 532 nm green diode laser through a fiber optic

as the excitation source and a 550 nm emission cutoff filter.

Crystallization plates containing PB-labeled ConA were

imaged using a 400 nm LED without an emission filter. The

screening plate created using a combination of two fluorescent

probes was imaged using both sets of optics. A single well

containing protein crystals was imaged under white light

(Fig. 4a), the CR optics (Fig. 4b) and the PB optics (Fig. 4c).

Other pairs of fluorescent probes may be employed for two-

color fluorescence. The ability to distinguish the fluorescence

of one probe in the presence of the other is essential. Costs

will be higher owing to the required emission filter(s). LEDs

typically have a full-width half-maximum wavelength spread

of��15�. There is still considerable excitation light relative to

the fluorescence signal at the �0.1–1% intensity level at the

peak fluorescence wavelength for probes with a short Stokes

shift. Use of probes with a longer Stokes shift, for example PB

and CY, can ameliorate this problem. The other approach, as

taken here, is to use a monochromatic light source. While this

adds to the complexity, diode lasers can be obtained online at

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Acta Cryst. (2017). F73, 657–663 Tarver & Pusey � A low-cost method for visible fluorescence imaging 661

Figure 4Two-color fluorescence imaging. (a) Protein crystals of ConA labeled with the fluorescent probes CR and PB under white light. (b) Image of the wellshown in (a) under the light of a 532 nm green laser diode using a 550 nm high-pass emission filter. (c) Image of the well shown in (a) illuminated with a400 nm blue LED with no emission filter.

a lower cost than that of emission filters. However, emission

filters are still sometimes needed to keep reflected light out of

the image.

3.5. Proteins

In order to examine this imaging method with additional

proteins, crystals of �-lactoglobulin B and trypsin were

photographed. �-Lactoglobulin B and trypsin crystals were

imaged under white light (Figs. 5a and 5c). The same

�-lactoglobulin B well was photographed again using the same

532 nm green diode laser, fiber optic and 550 nm emission

cutoff filter as used to image CR-labeled ConA crystals

(Fig. 5b). CY-labeled trypsin crystals were imaged using a

400 nm blue LED for excitation without an emission filter

(Fig. 5d).

3.6. Imaging

All fluorescent images were acquired using an iPhone 6S.

However, the use of this method is not limited to cell phones.

The clip-on lens can be applied to most devices with a camera,

but the camera has to be capable of accommodating the macro

optics. If image capture is not desired and an emission filter is

not needed, then one can bypass the use of a camera. Fluor-

escing crystals can be observed by manually placing the hood

with a mounted LED over a plate of TFL crystals and viewing

them under a low-powered microscope (a microscope that is

normally used to manually review crystallization plates).

Fluorescence from crystals at least as small as 10 mm can be

visualized in a dark room and, depending upon the microscopy

system, one can zoom in for higher magnification imaging. The

room does not have to be totally dark, but it is necessary to

remove reflected light from overhead fluorescent lights. In

fact, it was discovered that one can directly see CY-labeled

crystals without the use of a microscope. However, this is not a

practical approach when using probes that require near-UV

wavelengths for excitation. If the microscope has a camera

port then it can also be used for image capture. Low-cost

means of attaching a cell phone to microscopy or other

imaging systems are also commercially available.

This method was tested using several imaging devices. The

iPhone 6S contained the most versatile imaging system with

internal filters, and it provided the best quality images. Several

different macro lenses were tested, and the AUKEY Ora lens

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662 Tarver & Pusey � A low-cost method for visible fluorescence imaging Acta Cryst. (2017). F73, 657–663

Figure 5(a) Crystals of CR-labeled �-lactoglobulin B under white light. (b) The well shown in (a) imaged using a 532 nm green laser diode and a 550 nm high-passemission filter. (c) CY-labeled trypsin crystals under white light. (d) The well shown in (c) imaged under a 400 nm blue LED without an emission filter.

was found to perform the best at a low cost. The presence of

the hood served to reduce background illumination and it was

a convenient mounting point for the excitation light. Crystals

as small as 10 mm could be imaged, and possibly smaller with

the use of higher magnification optics. Other clip-on macro

lenses are commercially available and may be more suitable

for certain imaging devices.

While not tested, the method of Lukk et al. (2016) may be

accommodated by the approach described here. The apparent

Stokes shift for excitation at 405 nm is�80 nm. The excitation

source for their work was a 5 mW laser. If the added cost (and

complexity) is acceptable then the LED can be replaced by a

405 nm diode laser, bringing the light into the hood using a

fiber optic through the same aluminium tube as used for the

LED. However, not all proteins fluoresce. The advantage of

the TFL approach over other fluorescence methods is that the

fluorescence probe, and thus a signal, will be present. Previous

work has clearly shown that the presence of the probe at the

target labeling concentration range of 0.1–0.5% will not affect

the nucleation rates or quality of the diffraction data obtained

(Forsythe et al., 2006).

3.7. Costs

The cost of a simple system, composed of an imaging lens

and LED (Fig. 1) is less than $50. The cost can be less than $30

depending on the source of the LED, the power supply and

how the current-limiting resistor is implemented. The

approach taken can potentially be applied to UV fluorescence,

but at increased cost driven by the higher price of UV LEDs.

Other variations are possible, such as the use of low-cost diode

lasers for the excitation source, as demonstrated here for

imaging CR-labeled proteins. This approach involves a more

complex hardware setup as well as the higher cost of the laser

and fiber optic. An emission filter may be required to keep

reflected laser light noise out of the signal.

Most automated imaging systems are very expensive. The

method of Watts et al. (2010) is closest to our method in cost at

less than 1000 Euro. Their approach used the increase in the

fluorescence of the dye 1,8-ANS (excitation at �360 nm and

emission at �505 nm) upon diffusing into and binding to the

interior of a protein crystal (Groves et al., 2007). In their

implementation, the whole plate was imaged, with each well

being excited using a separate LED. The excitation source was

directed towards the imaging optics while utilizing both

excitation and emission filters. Their design also used

crystallization-plate-specific masks to limit the excitation light

to only the crystalline-drop positions. Software filtering was

used to reduce the background noise. As the method relies

upon free probe diffusing into the crystal, it cannot be used for

multiple colors. In comparison, the method presented here is a

low-cost approach for anyone owning a smart device with a

camera. It provides a way to rapidly screen plates containing a

wide variety of crystallization solutions and image protein

crystals. This approach can also be used for the imaging of

complexes, which can save beam time.

Several potential improvements immediately became

apparent using this method. When using an autofocusing

device, the camera must be securely fixed in position to more

easily obtain quality images. A Bluetooth shutter control and

tripod were later utilized to address this issue. While these

items did stabilize and improve the imaging system, they

increased the overall cost. Therefore, all images shown were

taken using a handheld iPhone 6S camera while resting the

lens hood on the crystallization plate. A second improvement

would be an application written for the smart device to adjust

the focal point and other exposure parameters, such as filters

that may be built into the camera.

Acknowledgements

The content is solely the responsibility of the authors and does

not necessarily represent the official views of the National

Institutes of Health.

Funding information

The research reported in this publication was supported by

National Institute of General Medical Sciences of the National

Institutes of Health under award No. R42GM116283.

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